The relative rates of phosphorylation for brain glucose measured from radioactively labeled 2-deoxyglucose and glucose were compared in this report. (1-14C) 2-Deoxyglucose and (6-3H)-glucose were simultaneously administered to brain via the internal carotid artery. The relative rates of glucose phosphorylation were determined from the 14C/3H ratios in the neutral and acidic metabolites in freeze-blown brain. The ratios of 14C/3H in the neutral metabolites were significantly elevated compared to that in the injection solution, indicating preferential uptake of (1-14C)-2-deoxyglucose over (6-3H)-glucose into brain. During the first minute after injection, the ratio of (1-14C)-2-deoxyglucose/(6-3H)-glucose increased. Subsequently, the ratio decreased, indicating that more tritium radioactivity from the phosphorylated product was returning to the brain glucose pool. The initial ratios of 14C/3H in the acidic metabolites were significantly less than that in the injection solution, indicating that the rate of phosphorylation for labeled glucose is greater than that of 2-deoxyglucose. The rate of glucose phosphorylation from labeled 2-deoxyglucose relative to that from labeled glucose, corrected for the dephosphorylation of hexose phosphates, was estimated to be 0.35. This value is about 25% less than that of the lumped constant which was determined from the "cerebral extraction" of glucose and 2-deoxyglucose. Since "cerebral extraction" reflects the balance between hexose phosphorylation and dephosphorylation, the difference between these two results suggest dephosphorylation of glucose-6-phosphate at 25% of the rate of glucose phosphorylation may have been inadvertently included in the estimation of the lumped constant by previous workers.